Antitumor agent

ABSTRACT

Pharmaceuticals which are effective for treatment, prevention, and the like of cancer and have less side effects are disclosed. The antitumor agent of the present invention comprises as an effective ingredient at least one leukotriene inhibitor. Examples of the leukotriene inhibitor includes leukotriene production inhibitors and leukotriene receptor antagonists, and preferred specific examples of the leukotriene inhibitor include montelukast, zafirlukast, pranlukast, and zileuton; pharmaceutically acceptable salts of these compounds; and pharmaceutically acceptable solvates of these compounds and the salts. The leukotriene inhibitor can also be used as a relieving agent for pain accompanying a tumor(s), and as a stromal hyperplasia inhibitor.

TECHNICAL FIELD

The present invention relates to an antitumor agent, a relieving agentfor pain accompanying a tumor(s), and a stromal hyperplasia inhibitor,comprising a leukotriene inhibitor as an effective ingredient.

BACKGROUND ART

Cancer is a disease that accounts for a large proportion of causes ofdeath in contemporary Japanese. A variety of anticancer agents have beendeveloped and practically used so far, but the anticancer agents havestrong side effects, and their burden to the bodies of patients islarge. In recent years, heavy particle radiotherapy has been practicallyused, but the cost of the therapy is high, and the therapy is availableonly in limited facilities. Thus, heavy particle radiotherapy cannot besaid to be a general option that can be freely selected at present.Therefore, anticancer agents which are highly effective against cancer,have only small side effects, and can be provided at low cost, are stillstrongly demanded.

As an example of a low-cost anticancer agent, an antitumor agentcontaining as an effective ingredient an antiallergic agent oxatomide orazelastine, or a pharmaceutically acceptable salt thereof, is disclosedin Patent Document 1. Both oxatomide and azelastine are agentsclassified as histamine H1 receptor antagonists. The antitumor uses ofoxatomide and azelastine and pharmaceutically acceptable salts thereofdescribed in Patent Document 1 are, more specifically, therapeutic usesfor malignant tumors. Patent Document 1 does not disclose at all thatleukotriene inhibitors such as leukotriene receptor antagonists areactually effective for various epithelial and nonepithelial tumors whichhave been spontaneously developed in vivo while producing few sideeffects. Patent Document 1 also does not disclose an effect on benigntumors. Moreover, Patent Document 1 does not at all describe actions ofoxatomide and azelastine on stromal components such as nerves, bloodvessels, and fibroblasts in tumor sites.

PRIOR ART DOCUMENT(S) Patent Document(s)

Patent Document 1: JP 2003-252763 A

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

An object of the present invention is to provide pharmaceuticals whichare effective for treatment, prevention, and the like of cancer and haveless side effects.

Means for Solving the Problems

As a result of intensive study, the present inventors discovered thatleukotriene inhibitors are effective for tumors spontaneously developedin vivo, and hardly produce side effects. Moreover, since degenerationof peripheral nerve cells in tumor sites was found in groups treatedwith a leukotriene inhibitor, leukotriene inhibitors were found to bealso effective for relieving pain accompanying tumors. In addition, thepresent inventors carried out immunostaining of tissue samples ofvarious human-derived tumors as well as spontaneously-developed rattumor tissues using an antibody against leukotriene receptors. As aresult, the presence of cells positive for the leukotriene receptorantibody could be found in all of the various tumor tissuesinvestigated, irrespective of whether the tumor is epithelial ornonepithelial and whether the tumor is malignant or benign. Thus, theleukotriene inhibitors have been confirmed to be effective for varioustumors, thereby completing the present invention.

That is, the present invention provides an antitumor agent comprising atleast one leukotriene inhibitor as an effective ingredient. The presentinvention also provides a relieving agent for pain accompanying atumor(s), which agent comprises at least one leukotriene inhibitor as aneffective ingredient. The present invention also provides a stromalhyperplasia inhibitor comprising at least one leukotriene inhibitor asan effective ingredient. The present invention also provides a methodfor treatment, prevention, metastasis suppression, or recurrencesuppression of a tumor(s) in a subject in need thereof, which methodcomprises administering an effective amount of at least one leukotrieneinhibitor to the subject. The present invention also provides a methodfor relieving pain accompanying a tumor(s) in a subject in need thereof,which method comprises administering an effective amount of at least oneleukotriene inhibitor to the subject. The present invention alsoprovides a method for inhibiting stromal hyperplasia in a subject inneed thereof, which method comprises administering an effective amountof at least one leukotriene inhibitor to the subject. The presentinvention also provides a leukotriene inhibitor for use in treatment,prevention, metastasis suppression, or recurrence suppression of atumor(s). The present invention also provides a leukotriene inhibitorfor use in relieving pain accompanying a tumor(s). The present inventionalso provides a leukotriene inhibitor for use in inhibiting stromalhyperplasia. The present invention also provides a method for screeningof an antitumor agent, a relieving agent for pain accompanying atumor(s), or a stromal hyperplasia inhibitor, which method uses as anindicator an ability to inhibit a signaling pathway mediated by bindingof leukotriene to a leukotriene receptor. The present invention alsoprovides a method for producing an antitumor agent, a relieving agentfor pain accompanying a tumor(s), or a stromal hyperplasia inhibitor,which method comprises the steps of: screening of an antitumor agent, arelieving agent for pain accompanying a tumor(s), or a stromalhyperplasia inhibitor by the screening method according to theabove-described present invention; and producing the antitumor agent,the relieving agent for pain accompanying a tumor(s), or the stromalhyperplasia inhibitor obtained by the screening.

EFFECT OF THE INVENTION

The antitumor agent of the present invention has been confirmed to beactually effective for tumors spontaneously developed in vivo, and tohardly produce side effects. Since leukotriene inhibitors practicallyused as antiallergic agents can be used as effective ingredients, theantitumor agent of the present invention can be provided at low cost.The present inventors revealed that leukotriene receptors are expressedin various tumors irrespective of whether the tumor is epithelial ornonepithelial, and whether malignant or benign (see Examples below).Leukotriene inhibitors can exert their antitumor effects against varioustumors by inhibiting signal transduction mediated by leukotrienereceptors in tumor sites by actions such as an antagonistic action onleukotriene receptors or inhibition of leukotriene production.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. A is an electron micrograph of a tumor tissue of the untreatedtumor-bearing rat group (Comparative Example 1). E, vascular endothelialcell; L, intravascular lumen; V, blood vessel; C, collagen fiber.

Fig. B is an electron micrograph of a tumor tissue of the tumor-bearingrat group with administration of montelukast sodium (Example 1; dosingperiod, 3 days). RBC, red blood cell; L, intravascular lumen; V, bloodvessel; C, collagen fiber; F, fibroblast; E-apo, apoptosis of vascularendothelial cell.

Fig. C is an electron micrograph of a tumor tissue of the tumor-bearingrat group with administration of montelukast sodium (Example 1; dosingperiod, 3 days). L, intravascular lumen; V, blood vessel; C, collagenfiber; F, fibroblast; E-apo, apoptosis of vascular endothelial cell;F-apo, apoptosis of fibroblast.

Fig. D is an electron micrograph of a tumor tissue of the tumor-bearingrat group with administration of pranlukast hydrate (Example 3; dosingperiod, 3 days). RBC, red blood cell; L, intravascular lumen; V, bloodvessel; C, collagen fiber; E-apo, apoptosis of vascular endothelialcell.

Fig. E is an electron micrograph of a tumor tissue of the tumor-bearingrat group with administration of pranlukast hydrate (Example 3; dosingperiod, 3 days). RBC, red blood cell; L, intravascular lumen; V, bloodvessel; C, collagen fiber; E-apo, apoptosis of vascular endothelialcell.

Fig. F is an electron micrograph of a tumor tissue of the tumor-bearingrat group with administration of pranlukast hydrate (Example 3; dosingperiod, 3 days). RBC, red blood cell; L, intravascular lumen; V, bloodvessel; C, collagen fiber; E-apo, apoptosis of vascular endothelialcell.

Fig. G is an electron micrograph of a tumor tissue of the tumor-bearingrat group with administration of zafirlukast (Example 2; dosing period,3 days). RBC, red blood cell; L, intravascular lumen; V, blood vessel;C, collagen fiber; E, vascular endothelial cell; F, fibroblast; E-apo,apoptosis of vascular endothelial cell; F-apo, apoptosis of fibroblast.

Fig. H is an electron micrograph of a tumor tissue of the tumor-bearingrat group with administration of zafirlukast (Example 2; dosing period,3 days). RBC, red blood cell; L, intravascular lumen; V, blood vessel;C, collagen fiber; F, fibroblast; E-apo, apoptosis of vascularendothelial cell.

Fig. I is an electron micrograph of a tumor tissue of the tumor-bearingrat group with administration of zafirlukast (Example 2; dosing period,3 days). L, intravascular lumen; V, blood vessel; C, collagen fiber; E,vascular endothelial cell.

Fig. J is an electron micrograph of a tumor tissue of the tumor-bearingrat group with administration of zileuton (Example 4; dosing period, 3days). RBC, red blood cell; L, intravascular lumen; V, blood vessel; C,collagen fiber; E-apo, apoptosis of vascular endothelial cell; F-apo,apoptosis of fibroblast.

Fig. K is an electron micrograph of a tumor tissue of the tumor-bearingrat group with administration of zileuton (Example 4; dosing period, 3days). RBC, red blood cell; L, intravascular lumen; V, blood vessel; C,collagen fiber; E-apo, apoptosis of vascular endothelial cell.

Fig. L is an electron micrograph of a smooth muscle cell in a tumor siteof the tumor-bearing rat group with administration of montelukast sodium(Example 1; dosing period, 3 days). SMC-Apo, apoptosis of a smoothmuscle cell.

Fig. M is an electron micrograph of a smooth muscle cell in a tumor siteof the tumor-bearing rat group with administration of pranlukast hydrate(Example 3; dosing period, 3 days). SMC-Apo, apoptosis of a smoothmuscle cell.

Fig. N is an electron micrograph of a smooth muscle cell in a tumor siteof the tumor-bearing rat group with administration of zafirlukast(Example 2; dosing period, 3 days). SMC-Apo, apoptosis of a smoothmuscle cell; C, collagen fiber.

Fig. O is an electron micrograph of a smooth muscle cell in a tumor siteof the tumor-bearing rat group with administration of zileuton (Example4; dosing period, 3 days). SMC-Apo, apoptosis of a smooth muscle cell.

Fig. P is an electron micrograph of a peripheral nerve cell in a tumorsite of the tumor-bearing rat group with administration of montelukastsodium (Example 1; dosing period, 3 days). a, axon; m, myelin sheath; ★,vacuolation and degeneration of an axon; arrow and , degeneration of amyelin sheath.

Fig. Q is an electron micrograph of a peripheral nerve cell in a tumorsite of the tumor-bearing rat group with administration of pranlukasthydrate (Example 3; dosing period, 3 days). a, axon; m, myelin sheath;★, vacuolation and degeneration of an axon; arrow and , degeneration ofa myelin sheath.

Fig. R is an electron micrograph of a peripheral nerve cell in a tumorsite of the tumor-bearing rat group with administration of zafirlukast(Example 2; dosing period, 3 days). a, axon; m, myelin sheath; ★,vacuolation and degeneration of an axon; arrow and , degeneration of amyelin sheath.

Fig. S is an electron micrograph of a peripheral nerve cell in a tumorsite of the tumor-bearing rat group with administration of zileuton(Example 4; dosing period, 3 days). a, axon; m, myelin sheath; ★,vacuolation and degeneration of an axon; arrow, degeneration of a myelinsheath.

Fig. T is an immunostaining image of a spontaneously-developed ratmammary gland tumor tissue, which image was obtained with a leukotrienereceptor antibody. Arrow, cell positively stained with the leukotrienereceptor antibody (cell stained brown).

Fig. U is an immunostaining image of a human breast cancer tissue, whichimage was obtained with a leukotriene receptor antibody. Arrow, cellpositively stained with the leukotriene receptor antibody (cell stainedbrown).

Fig. V is an immunostaining image of a human stomach cancer tissue,which image was obtained with a leukotriene receptor antibody. Arrow,cell positively stained with the leukotriene receptor antibody (cellstained brown).

Fig. W is an immunostaining image of a human uterine leiomyoma, whichimage was obtained with a leukotriene receptor antibody. Arrow, cellpositively stained with the leukotriene receptor antibody (cell stainedbrown).

Fig. X is an immunostaining image of a human leiomyosarcoma, which imagewas obtained with a leukotriene receptor antibody. Arrow, cellpositively stained with the leukotriene receptor antibody (cell stainedbrown).

MODE FOR CARRYING OUT THE INVENTION

In the present invention, the “leukotriene inhibitor” is a substancethat inhibits a signaling pathway mediated by binding of leukotriene toa leukotriene receptor. The inhibition may occur in any step along thepathways from biosynthesis of leukotriene to the signaling pathwayslocated downstream of the leukotriene receptor.

Possible specific examples of the leukotriene inhibitor includesubstances that inhibit production of leukotriene, substances thatinhibit binding of leukotriene to a leukotriene receptor, and substancesthat suppress activation of leukotriene receptors to inhibit signaltransduction to the downstream factors. Typically, the leukotrieneinhibitor may be a substance that inhibits or suppresses a function ofleukotriene in vivo, and includes leukotriene production inhibitors andleukotriene receptor antagonists.

In the present invention, examples of the “leukotriene productioninhibitors” include substances that inhibit factors such as enzymes thatfunction in a leukotriene biosynthetic pathway (leukotriene biosynthesisinhibitors), and substances that inhibit release of leukotriene fromcells which produce leukotriene, such as mast cells, eosinophils,neutrophils, basophils, and monocytes (leukotriene release inhibitors).Specific examples of leukotriene production inhibitors that may be usedin the present invention include 5-lipoxygenase inhibitors such aszileuton, ABT-761, CJ-13610, ZD-2138, AA-861, and cirsiliol; inhibitorsof 5-lipoxygenase-activating proteins, such as MK-886 and BAY X1005.Pharmaceutically acceptable salts of these compounds, andpharmaceutically acceptable solvates of these compounds and saltsthereof may also be used. However, the leukotriene production inhibitorsthat may be used as effective ingredients in the present invention arenot limited to these specific examples.

In the present invention, the “leukotriene receptor antagonists” includevarious substances that antagonistically function against the action ofleukotriene on a leukotriene receptor. Examples of the leukotrienereceptor antagonists include substances that competitively ornoncompetitively inhibit binding of leukotriene to a leukotrienereceptor, and substances having an action to suppress activation of aleukotriene receptor, to inhibit signal transduction to the downstreamfactors. Examples of the “leukotriene receptor antagonists” also includemolecules such as inverse agonists which bind to a leukotriene receptorto stabilize the receptor into an inactive structure, which are oneexample of antagonists. Specific examples of the leukotriene receptorantagonists that may be used in the present invention includezafirlukast, montelukast, pranlukast, FPL55712, BAYu9773, LY293111 Na,CGS 25019C, ONO-4057, SB201993, CP195543, SC53228, and BIIL 284 (BIIL260). Pharmaceutically acceptable salts of these compounds, andpharmaceutically acceptable solvates of these compounds and saltsthereof may also be used. However, the leukotriene receptor antagoniststhat may be used as effective ingredients in the present invention arenot limited to these specific examples.

Examples of the pharmaceutically acceptable salts include acid additionsalts such as hydrochloric acid salt, sulfuric acid salt, fumaric acidsalt, maleic acid salt, tartaric acid salt, and citric acid salt. Incases where the leukotriene inhibitor is an acid, examples of thepharmaceutically acceptable salts also include metal salts such assodium salt, potassium salt, and calcium salt. Examples of thepharmaceutically acceptable solvates include solvates with an organicsolvent, and hydrates. However, the pharmaceutically acceptable saltsand the pharmaceutically acceptable solvates are not limited to thesespecific examples.

Preferred specific examples of the leukotriene inhibitor include atleast one selected from the group consisting of: compounds selected frommontelukast, zafirlukast, pranlukast, and zileuton; pharmaceuticallyacceptable salts of the compounds; and pharmaceutically acceptablesolvates of the compounds and the salts of the compounds.

In the present invention, the term “antitumor” includes suppression oftumorigenesis (initiation, metastasis, and recurrence) and suppressionof tumor proliferation. Accordingly, the “antitumor agent” includestherapeutic agents, preventive agents, metastasis-suppressing agents,and recurrence-suppressing agents for tumors. The term “tumor” includesboth benign tumors and malignant tumors. More specifically, the tumorcan be classified into malignant epithelial tumors, benign epithelialtumors, malignant nonepithelial tumors, and benign nonepithelial tumors.Examples of the malignant tumors (cancers) include solid cancers andblood cancers. Examples of the solid cancers include carcinomas(malignant epithelial tumors), sarcomas (malignant nonepithelialtumors), and nervous system malignant solid tumors such as melanoma andglioma (malignant nonepithelial tumors). Examples of the blood cancersinclude blood malignant tumors such as leukemia, malignant lymphoma, andmultiple myeloma. The term “solid tumor” includes not only solidcancers, but also benign solid tumors.

The antitumor agent of the present invention comprises at least oneleukotriene inhibitor as an effective ingredient. The antitumor agentmay comprise two or more leukotriene inhibitors as effectiveingredients. In cases where the antitumor agent of the present inventionis used for treatment of malignant tumors, agents containing as aneffective ingredient at least one selected from oxatomide and azelastineand pharmaceutically acceptable salts thereof may be excluded from theantitumor agent of the present invention irrespective of whether or notthese antiallergic agents are included in the leukotriene inhibitordefined in the present invention. In cases where the antitumor agent ofthe present invention is used as a metastasis-suppressing agent or arecurrence-suppressing agent for malignant tumors, agents comprising asan effective ingredient at least one selected from oxatomide andazelastine and pharmaceutically acceptable salts thereof may be excludedfrom the antitumor agent of the present invention. In cases where theantitumor agent of the present invention is used as a preventive agentfor malignant tumors, agents comprising as an effective ingredient atleast one selected from oxatomide and azelastine and pharmaceuticallyacceptable salts thereof may be excluded from the antitumor agent of thepresent invention.

As described in Examples below, expression of leukotriene receptors hasbeen confirmed in various types of tumors irrespective of whether thetumor is benign or malignant, and whether epithelial or nonepithelial.Leukotriene inhibitors are thought to exert their antitumor effects byinhibiting signal transduction mediated by leukotriene receptorsexpressed in the tumor site, by an antagonistic action against theleukotriene receptors or by inhibition of leukotriene production. Thus,leukotriene inhibitors can exert their antitumor effect widely againstmalignant and benign epithelial tumors, and malignant and benignnonepithelial tumors. The antitumor agent of the present invention canbe preferably used for, e.g., solid tumors, or malignant tumors, orsolid cancers, although uses of the antitumor agent are not limitedthereto.

Leukotriene inhibitors have an action to induce apoptosis of tumor cellsspontaneously developed in vivo (see Examples below). In particular,since apoptosis and degeneration are also found not only in endothelialcells of newly formed blood vessels, which are stromal components oftumor tissues and indispensable as routes for supplying enzymes andnutrients to tumor cells, but also in other stromal cells such asperipheral nerve cells that proliferate side by side with newly formedblood vessels in tumor sites and smooth muscle cells involved in thecontractile motion of blood vessels, leukotriene inhibitors can bepreferably used for solid tumors, especially solid cancers, amongtumors.

Induction of apoptosis of endothelial cells of newly formed bloodvessels by a leukotriene inhibitor is found only in blood vessels insolid tumor tissues, and not found in non-tumor healthy tissues. Fromthe viewpoint of this effect, the antitumor agent of the presentinvention used for solid tumors can also be said to be an angiogenesisinhibitor specific to solid tumors. From the viewpoint of the inhibitoryaction against hyperplasia of stromal cells, the agent can also be saidto be a stromal hyperplasia inhibitor specific to solid tumors. Newlyformed blood vessels are indispensable as routes for supplying enzymesand nutrients to tumor cells, and other stromal components such asfibroblasts, collagen fibers produced by fibroblasts, peripheral nervecells, and smooth muscle cells constitute a tumor site together with thetumor cells. Since a leukotriene inhibitor can suppress the growth andmetastasis of a solid tumor by blocking the routes for supplying enzymesand nutrients to tumor cells and killing proliferated stromal cells,treatment and prevention of solid tumors (including prevention ofmetastasis and recurrence of malignant tumors) are possible.

However, since, as described above, induction of apoptosis by aleukotriene inhibitor can be found not only for newly formed bloodvessels, but also for tumor cells themselves, the effect of theantitumor agent of the present invention is not limited to solid tumors,and the agent is also effective for blood cancers.

In general, in cases where a patient is judged as being suspected ofhaving a cancer in medical examination or health screening, and then isfound to have a benign tumor as a result of detailed examination, thepatient is followed up thereafter by regularly visiting a hospital forthe purpose of early detection and treatment of malignant change(canceration). Since leukotriene inhibitors exert their therapeuticeffects such as growth inhibition also on benign tumors, leukotrieneinhibitors are also useful for prevention of tumors (more specifically,malignant tumors). In cases where the antitumor agent is used for thepurpose of prevention, the agent may be typically administered to asubject having a benign tumor, more specifically, a subject having abenign tumor which may undergo malignant change.

Since leukotriene inhibitors cause degeneration of peripheral nervecells in tumor sites and kill proliferated nerve cells in the tumorsites, leukotriene inhibitors are also effective for relieving painaccompanying tumors.

As described above, various leukotriene inhibitors are known, andmethods for synthesizing these leukotriene inhibitors are also known.

The administration route of the antitumor agent of the present inventionmay be oral administration or parenteral administration (e.g.intravenous administration, subcutaneous administration, intramuscularadministration, rectal administration or the like), and may be systemicadministration or topical administration. Oral administration ispreferred from the viewpoint of simplicity. The dose/dosage isappropriately set depending on the severity of symptoms (for example,the tumor size, the degree of progression, and/or the malignancy) in thepatient, the type of the leukotriene inhibitor used, and the like.Although an antitumor effect can be obtained at a dose/dosage at whichthe agent is used as an antiallergic agent, the antitumor agent may beadministered, if necessary, at a dose/dosage 10 to 100 times higher thanthat for use as an antiallergic agent, or at an even higher dose/dosage.The antitumor agent of the present invention may be used at an amount ofabout 1 mg to 200,000 mg, for example, about 10 mg to 100,000 mg, about100 mg to 100,000 mg, about 10 mg to 50,000 mg, about 100 mg to 50,000mg, or about 10 mg to 10,000 mg in terms of the amount of theleukotriene inhibitor per adult per day, although the dose/dosage is notlimited to these.

The dosage form of the antitumor agent is not limited, and may beappropriately selected depending on the administration route. Theantitumor agent can be produced by formulation techniques commonly usedin the field of pharmaceuticals. The antitumor agent of the presentinvention may have the same constitution as that of a formulationcommercially available as an antiallergic agent.

The subject to which the leukotriene inhibitor is administered is amammal, and examples of the mammal include, but are not limited to,human, dog, cat, rabbit, hamster, mouse, rat, ferret, horse, cattle,pig, sheep, and monkey.

By using as an indicator the ability to inhibit a signaling pathwaymediated by binding of leukotriene to a leukotriene receptor, screeningof a novel antitumor agent, a relieving agent for pain accompanying atumor(s), or a stromal hyperplasia inhibitor can be carried out. Theinhibition of a signaling pathway mediated by binding of leukotriene toa leukotriene receptor may be, as described above, inhibition ofleukotriene production, inhibition of binding of leukotriene to aleukotriene receptor, suppression of activation of a leukotrienereceptor to inhibit signal transduction to the downstream factors, orthe like. Whether or not an agent has an action to inhibit the signalingpathway can be investigated based on, for example, whether or not theagent inhibits the function of a promotional factor in the signalingpathway, or whether or not the agent promotes the function of asuppressive factor in the signaling pathway. The inhibition of thefunction of the promotional factor may be, for example, inhibition ofexpression of the factor in the cell, or inhibition of a physiologicalactivity of the factor. The promotion of the function of the suppressivefactor may be, for example, promotion of expression of the factor in thecell, or promotion of a physiological activity of the factor.

EXAMPLES

The present invention is concretely described below by way of Examples.However, the present invention is not limited to the Examples below.

<Materials>

Sprague-Dawley rats which had spontaneously developed a tumor duringbreeding (hereinafter referred to as tumor-bearing rats) were purchasedfrom Charles

River Japan, Inc., and used for experiments. Tumors observed were allmammary gland tumors (adenocarcinoma and adenoma). All of these werespontaneous tumors, and the rats were cancer-bearing animals havingvarious conditions of tumorigenesis including carcinogenic factors inmammals including human.

<Methods> Comparative Example 1 Observation of Tumor Lesion Samples fromUntreated Tumor-Bearing Rats (3 Cases)

Under ether anesthetization, tumor tissues were removed fromtumor-bearing rats. The removed tumors were fixed, and thereaftersamples for light microscopy (hematoxylin-eosin staining) and samplesfor electron microscopy (uranium-lead double staining) were prepared andobserved.

Example 1 Therapeutic Effect of Montelukast Sodium on Tumor-bearing Rats(3 Cases)

To tumor-bearing rats, a leukotriene inhibitor containing montelukastsodium as an active ingredient (leukotriene receptor antagonist; tradename “Singulair Tablets”; manufactured by MSD K. K.) was orallyadministered for up to 7 days at a daily dose of 0.16 mg/kg body weightin terms of montelukast sodium. On Day 3 and Day 7, tumor tissues wereremoved under ether anesthetization. In the same manner as inComparative Example 1, the removed tumors were fixed, and thereaftersamples for light microscopy (hematoxylin-eosin staining) and samplesfor electron microscopy (uranium-lead double staining) were prepared andobserved.

Example 2 Therapeutic Effect of Zafirlukast on Tumor-bearing Rats (3Cases)

To tumor-bearing rats, a leukotriene inhibitor containing zafirlukast asan active ingredient (leukotriene receptor antagonist; trade name“Accolate”;

manufactured by AstraZeneca K. K.) was orally administered for up to 7days at a daily dose of 1.33 mg/kg body weight in terms of zafirlukast.On Day 3 and Day 7, tumor tissues were removed under etheranesthetization. In the same manner as in Comparative Example 1, theremoved tumors were fixed, and thereafter samples for light microscopy(hematoxylin-eosin staining) and samples for electron microscopy(uranium-lead double staining) were prepared and observed.

Example 3 Therapeutic Effect of Pranlukast Hydrate on Tumor-Bearing Rats(3 Cases)

To tumor-bearing rats, a leukotriene inhibitor containing pranlukasthydrate as an active ingredient (leukotriene receptor antagonist; tradename “Onon”; manufactured by Ono Pharmaceutical Co., Ltd.) was orallyadministered for up to 7 days at a daily dose of 7.5 mg/kg body weightin terms of pranlukast hydrate. On Day 3 and Day 7, tumor tissues wereremoved under ether anesthetization. In the same manner as inComparative Example 1, the removed tumors were fixed, and thereaftersamples for light microscopy (hematoxylin-eosin staining) and samplesfor electron microscopy (uranium-lead double staining) were prepared andobserved.

Example 4 Therapeutic Effect of Zileuton on Tumor-Bearing Rats (3 Cases)

To tumor-bearing rats, a leukotriene inhibitor containing zileuton as anactive ingredient (leukotriene biosynthesis inhibitor; trade name“ZYFLO”; manufactured by Abbott Laboratories) was orally administeredfor up to 7 days at a daily dose of 34.3 mg/kg body weight in terms ofzileuton. On Day 3 and Day 7, tumor tissues were removed under etheranesthetization. In the same manner as in Comparative Example 1, theremoved tumors were fixed, and thereafter samples for light microscopy(hematoxylin-eosin staining) and samples for electron microscopy(uranium-lead double staining) were prepared and observed.

<Results>

Unlike the tumor tissue images of the untreated tumor-bearing rats ofComparative Example 1, in the tumor cells in all tumor-bearing ratstreated with a leukotriene inhibitor, that is, montelukast sodium(leukotriene receptor antagonist, Example 1), zafirlukast (leukotrienereceptor antagonist, Example 2), pranlukast hydrate (leukotrienereceptor antagonist, Example 3), or zileuton (leukotriene biosynthesisinhibitor, Example 4), apoptosis was already induced on as early as Day3, and a remarkable inhibitory effect on the development and the growthof the tumor cells was observed.

In all the treated groups, apoptosis was induced in endothelial cells ofnewly formed blood vessels, which are stromal components of tumortissues and indispensable as routes for supplying enzymes and nutrientsto tumor cells. It was therefore thought that blood flow in the tumorwas blocked, leading to the inhibitory effect on the development and thegrowth of the tumor cells. In addition to vascular endothelial cells,apoptosis was also observed in fibroblasts and smooth muscle cells, andmoreover degeneration of peripheral nerve cells extending side by sidewith blood vessels was also found. Such apoptosis and degeneration ofstromal cells were found only in tumor tissues in the treated groups,and not found in normal sites in all treated groups and tumor tissues inthe untreated group.

No side effects were observed in the rats in the treated groups.

The pathological findings described above are explained below in moredetail based on micrographs of pathological specimens.

Fig. A is an electron micrograph of a tumor tissue of the untreatedgroup (Comparative Example 1), wherein a normal vascular endothelialcell (E in the figure) is observed.

Figs. B and C are electron micrographs of tumor tissues of the groupwith administration of montelukast sodium (Example 1, dosing period, 3days). In blood vessels in the tumor tissues, apoptosis of endothelialcells (E-apo in the figures) was observed. These endothelial cellsshowed irregular condensation and fragmentation (* in the figures) oftheir nuclei. These conditions correspond to Stage II to III ofapoptosis (T. Ihara, et al. The process of ultrastructural changes fromnuclei to apoptotic body. Virchow Arch (1998) 433: 443-447). By theapoptosis of endothelial cells, an effect to inhibit blood flow into thetumor tissues (an inhibitory effect on newly formed blood vessels) couldbe obtained. Apoptosis of fibroblasts (F-apo in the figures), whichproduces collagen fibers and constitutes the stroma of the tumor tissue,was induced, and reduction in collagen fibers (C in the figures) wasalso observed. Thus, an effect to inhibit stromal hyperplasia was alsoseen.

Figs. D to F are electron micrographs of tumor tissues of the group withadministration of pranlukast hydrate (Example 3; dosing period, 3 days).In blood vessels in the tumor tissues, apoptosis of endothelial cells(E-apo in the figures) was observed. These endothelial cells showedirregular condensation and fragmentation (* in the figures) of theirnuclei. These conditions correspond to Stage II to III of apoptosis (T.Ihara, et al. 1998, described above). Similarly to the group withadministration of zileuton, an effect to inhibit blood flow into thetumor tissues by apoptosis of endothelial cells (an inhibitory effect onnewly formed blood vessels) could be obtained.

Figs. G to I are electron micrographs of tumor tissues of the group withadministration of zafirlukast (Example 2; dosing period, 3 days). Inblood vessels in the tumor tissues, apoptosis of endothelial cells(E-apo in the figures) was observed. These endothelial cells showedirregular condensation and fragmentation (* in the figures) of theirnuclei. These conditions correspond to Stage II to III of apoptosis (T.Ihara, et al. 1998, described above). In addition, degeneration (→) ofvascular endothelial cells (E) was observed (Fig. I). By the apoptosisand the degeneration of endothelial cells, an effect to inhibit bloodflow into the tumor tissues (an inhibitory effect on newly formed bloodvessels) could be obtained.

Figs. J and K are electron micrographs of tumor tissues of the groupwith administration of zileuton (Example 4, dosing period, 3 days). Inblood vessels in the tumor tissues, apoptosis of endothelial cells(E-apo in the figures) was observed. These endothelial cells showedirregular condensation and fragmentation (* in the figures) of theirnuclei. These conditions correspond to Stage II to III of apoptosis (T.Ihara, et al. 1998, described above). By the apoptosis of endothelialcells, an effect to inhibit blood flow into the tumor tissues (aninhibitory effect on newly formed blood vessels) could be obtained.Apoptosis of fibroblasts (F-apo in the figures), which produce collagenfibers and constitute the stroma of the tumor tissues, was also induced.Thus, an effect to inhibit stromal hyperplasia was also seen.

Figs. L to O are electron micrographs of smooth muscle cells of thegroups with administration of leukotriene inhibitors. In any of thetreated groups, apoptosis of smooth muscle cells (SMC-Apo) in the tumortissue was observed. These smooth muscle cells showed irregularcondensation and fragmentation (*) of their groups. These conditionscorrespond to Stage II to III of apoptosis (T. Ihara, et al. 1998,described above). Due to the apoptosis of smooth muscle cells in thetumor tissues, an inhibitory effect on newly formed blood vessels(because apoptosis was also observed in smooth muscle cells in thevicinities of blood vessels) and an inhibitory effect on stromalhyperplasia (because smooth muscle cells, together with fibroblasts,produce collagen fibers, which are components constituting the stroma)were found. These findings were found only in the leukotriene inhibitoradministration groups, and not found in the untreated group.

Figs. P to S are electron micrographs of peripheral nerve cells of thegroups with administration of leukotriene inhibitors. In peripheralnerves in the tumor tissues, degeneration of axons (a) and myelinsheaths (m) were observed. These findings indicate damage of theperipheral nerve cells. Thus, the administration of the leukotrieneinhibitors produced an effect to suppress proliferation of nerves thatextend side by side with blood vessels. In addition, an inhibitoryeffect on tumor pain can be obtained because of the suppression of nervecell proliferation in the tumor tissue. These findings were found onlyin the leukotriene inhibitor administration groups, and not found in theuntreated group. The degeneration was not observed at all in peripheralnerve cells in the contralateral side of the tumor site (non-tumor site)of each tumor-bearing rat in the groups with administration ofleukotriene inhibitors. Therefore, the leukotriene inhibitors werethought to have no side effects.

Example 5 Investigation of Leukotriene Receptors by Immunostaining UsingLeukotriene Receptor Antibody

Whether leukotriene receptors are present or not was investigated forvarious tumor tissues using their paraffin sections, by animmunostaining method (Simple Stain MAX PO method, Nichirei Corporation)using leukotriene receptor antibodies (Polyclonal Antibody to CysLT1 andPolyclonal Antibody to CysLT2, Acris Antibodies GmbH). The tumor tissuesinvestigated are shown in Table 1.

TABLE 1 Rat Spontaneous mammary gland tumor Human (Malignant stomachcancer, colon cancer, liver cancer, renal tumors) cancer, thyroidcancer, pharyngeal cancer, esophageal cancer, bile duct cancer,pancreatic cancer, breast cancer, prostate cancer, leiomyosarcoma,rhabdomyosarcoma, angiosarcoma (mammary gland) (Benign pituitaryadenoma, meningioma, goiter, tumors) neurilemmoma, leiomyoma

In all tumor tissues investigated in the present Example, cells positivefor the leukotriene receptor antibody could be found. Representativeexamples of the immunostaining images are shown in Figs. T to X.

The four kinds of leukotriene inhibitors used above are drugs whichantagonistically act against leukotriene receptors or which inhibitproduction of leukotriene. Since a large number of leukotriene receptorsare expressed in various cells in the tumor tissues, it is thought thatthe therapeutic effects against tumors are obtained by inhibition ofsignal transduction mediated by leukotriene receptors. Since expressionof leukotriene receptors is observed in both epithelial andnonepithelial tumor tissues, leukotriene inhibitors are thought to exerttheir therapeutic effects in both tumor groups. Moreover, sinceexpression of leukotriene receptors was also confirmed in benign tumortissues, leukotriene inhibitors are thought to exert their therapeuticeffects not only in malignant tumors, but also in benign tumors.

1-19. (canceled)
 20. A method for treatment or prevention of a malignanttumor(s), excluding suppression of tumor metastasis, in a subject inneed thereof, said method comprising administering to said subject aneffective amount of at least one leukotriene inhibitor selected from thegroup consisting of: compounds selected from montelukast, zafirlukast,and pranlukast; pharmaceutically acceptable salts of said compounds; andpharmaceutically acceptable solvates of said compounds and said salts.21. The method according to claim 20, wherein said tumor is a solidtumor(s).
 22. The method according to claim 20, which is a method forprevention of a malignant tumor(s).
 23. The method according to claim21, which is a method for prevention of a malignant tumor(s).